Nanobody-glycan modifying enzyme fusion proteins and uses thereof

Abstract

The present disclosure provides fusion proteins comprising a nanobody and a glycan modifying enzyme. Also provided herein are methods of glycosylating a protein and methods of removing a sugar from a protein. Further provided in the present disclosure are methods of treating and/or diagnosing diseases. Also provided herein are kits, polynucleotides, vectors, and cells.

What is claimed is: 
     
         1 . A fusion protein comprising (i) a nanobody, and (ii) a glycan modifying enzyme. 
     
     
         2 . The fusion protein of  claim 1 , wherein the nanobody is fused to the N-terminal domain of the enzyme. 
     
     
         3 . The fusion protein of  claim 1 , wherein the nanobody is fused to the C-terminus of the enzyme. 
     
     
         4 . The fusion protein of  claim 1 , wherein the enzyme is a glycosyl transferase. 
     
     
         5 . The fusion protein of  claim 4 , wherein the enzyme is O-GlcNAc transferase (OGT). 
     
     
         6 . The fusion protein of  claim 1 , wherein the enzyme is a glycosyl hydrolase. 
     
     
         7 . The fusion protein of  claim 6 , wherein the enzyme is O-GlcNAcase (OGA). 
     
     
         8 . The fusion protein of  claim 5 , wherein the enzyme comprises (i) a catalytic domain, and optionally, (ii) a tetratricopeptide repeat (TPR) domain. 
     
     
         9 . The fusion protein of  claim 8 , wherein the number of tetratricopeptide repeat (TPR) domains is selected from a group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. 
     
     
         10 . The fusion protein of any one of  claims 1 - 9 , wherein the nanobody binds a cell surface protein. 
     
     
         11 . The fusion protein of any one of  claims 1 - 9 , wherein the nanobody binds a green fluorescent protein (GFP). 
     
     
         12 . The fusion protein of  claim 1 , wherein the nanobody binds a specific peptide tag. 
     
     
         13 . The fusion protein of  claim 12 , wherein the specific peptide tag is a four-amino acid tag. 
     
     
         14 . The fusion protein of  claim 13 , wherein the four-amino acid tag is EPEA. 
     
     
         15 . The fusion protein of  claim 14 , wherein the nanobody binds the four-amino acid EPEA tag (nEPEA). 
     
     
         16 . The fusion protein of any one of  claims 1 - 9 , wherein the nanobody binds beta-catenin. 
     
     
         17 . The fusion protein of any one of  claims 1 - 9 , wherein the nanobody binds TET3. 
     
     
         18 . The fusion protein of any one of  claims 1 - 9 , wherein the nanobody binds alpha-synuclein. 
     
     
         19 . The fusion protein of any one of  claims 1 - 9 , wherein the nanobody binds Tau. 
     
     
         20 . The fusion protein of any one of  claims 1  to  19 , wherein the nanobody and the glycan modifying enzyme are fused via a linker. 
     
     
         21 . A method of glycosylating a protein, the method comprising contacting a target protein with a fusion protein of any one of  claims 1  to  20 . 
     
     
         22 . The method of  claim 21 , wherein the target protein is selected from the group consisting of nuclear proteins, cytosolic proteins, and mitochondrial proteins. 
     
     
         23 . The method of  claim 21 , wherein the target protein is selected from the group consisting of transcription factors, kinases, oxidoreductases, nucleoporins, and nuleosomes. 
     
     
         24 . The method of  claim 23 , wherein the transcription factor is selected from the group consisting of c-JUN, JUNB, IKZF1, and STAT1. 
     
     
         25 . The method of  claim 23 , wherein the kinase is Zap70. 
     
     
         26 . The method of  claim 23 , wherein the oxidoreductase is TET3. 
     
     
         27 . The method of  claim 23 , wherein the nucleoporin is selected from the group consisting of Nup35 and Nup62. 
     
     
         28 . The method of  claim 23 , wherein the nucleosome is selected from the group consisting of H2B, H3, and H4. 
     
     
         29 . A method of glycosylating a protein, the method comprising:
 contacting a target protein with a fusion protein of any one of  claims 1  to  20  in the presence of a glycosyl donor molecule, thereby installing the sugar moiety from the glycosyl donor molecule on the target protein.   
     
     
         30 . A method of glycosylating a protein, the method comprising:
 contacting a target protein with a fusion protein of any one of  claims 1  to  20  in the presence of a O-linked N-acetyl glucosamine donor molecule, thereby installing a O-linked N-acetyl glucosamine on the target protein via the addition of a glucosamine monosaccharide attached to serine or threonine.   
     
     
         31 . The method according to  claim 29  or  30 , wherein the target protein is alpha-synuclein. 
     
     
         32 . The method according to  claim 29  or  30 , wherein the target protein is Tau. 
     
     
         33 . The method according to  claim 29  or  30 , wherein the target protein is TET3. 
     
     
         34 . The method according to  claim 29  or  30 , wherein the target protein is beta-catenin. 
     
     
         35 . A method of removing a sugar from a protein, the method comprising:
 contacting a target protein containing a sugar with a fusion protein of any one of  claims 1  to  20 , thereby excising a sugar moiety from the target protein.   
     
     
         36 . A method of removing a sugar from a protein, the method comprising:
 contacting a target protein containing an O-linked N-acetyl glucosamine with a fusion protein of any one of  claims 1  to  20 , thereby excising an O-linked N-acetyl glucosamine from a serine or threonine residue of the target protein.   
     
     
         37 . The method according to  claim 35  or  36 , wherein the target protein is alpha-synuclein. 
     
     
         38 . The method according to  claim 35  or  36 , wherein the target protein is Tau. 
     
     
         39 . A method of studying the effect of glycosylation in a cell using the fusion protein of any one of  claims 1  to  20 . 
     
     
         40 . A method of treating a disease, the method comprising administering a fusion protein of any one of  claims 1  to  20  to a subject in need thereof. 
     
     
         41 . A method of diagnosing a subject with a disease, the method comprising administering a fusion protein of any one of  claims 1  to  20  to a subject. 
     
     
         42 . A method of treating a subject suffering from or susceptible to a neurodegenerative disease, the method comprising administering an effective amount of the fusion protein of any one of  claims 1  to  20 . 
     
     
         43 . The method of  claim 42 , wherein the neurodegenerative disease is selected from the group consisting of Parkinson's disease, Huntington's disease, Alzheimer's disease, dementia, and multiple system atrophy. 
     
     
         44 . The method according to  claim 43 , wherein the neurodegenerative disease is Parkinson's disease. 
     
     
         45 . The method according to  claim 43 , wherein the neurodegenerative disease is Huntington's disease. 
     
     
         46 . A method of treating a subject suffering from or susceptible to a psychotic disorder, the method comprising administering an effective amount of the fusion protein of any one of  claims 1  to  20 . 
     
     
         47 . The method of  claim 46 , wherein the psychotic disorder is schizophrenia. 
     
     
         48 . A method of treating a subject suffering from or susceptible to epilepsy, the method comprising administering an effective amount of the fusion protein of any one of  claims 1  to  20 . 
     
     
         49 . A method of treating a subject suffering from or susceptible to a sleep disorder, the method comprising administering an effective amount of the fusion protein of any one of  claims 1  to  20 . 
     
     
         50 . A method of treating a subject suffering from or susceptible to an addiction, the method comprising administering an effective amount of the fusion protein of any one of  claims 1  to  20 . 
     
     
         51 . A pharmaceutical composition comprising a compound of any one of  claims 1  to  20 , and a pharmaceutically acceptable excipient. 
     
     
         52 . A kit comprising:
 (a) a fusion protein of any one of  claims 1  to  20 ; and   (b) either a glycosyl donor molecule or a glycosyl acceptor molecule.   
     
     
         53 . The kit according to  claim 52 , wherein the glycosyl donor molecule is uridine diphosphate N-acetylglucosamine. 
     
     
         54 . A kit comprising:
 (a) a vector for expressing a fusion protein of any one of  claims 1  to  20 ; and   (b) either a glycosyl donor molecule or a glycosyl acceptor molecule.   
     
     
         55 . The kit according to  claim 54 , wherein the glycosyl donor molecule is uridine diphosphate N-acetylglucosamine. 
     
     
         56 . A polynucleotide encoding the fusion protein of any one of  claims 1  to  20 . 
     
     
         57 . A vector comprising the polynucleotide of  claim 56 . 
     
     
         58 . A cell comprising the fusion protein of any one of  claims 1  to  20 . 
     
     
         59 . A cell comprising the nucleic acid molecule encoding the fusion protein of any one of  claims 1  to  20 .